Antisense oligonucleotide technology presents an exciting new therapy for many diseases, including pathogenic infections, cancer, and inherited conditions. The field has progressed enormously over the past decade, and currently numerous clinical trials are in progress or are proposed. Antisense oligonucleotides act by binding to a target nucleic acid by Watson-Crick or Hoogstein base-pairing. Antisense oligonucleotides may be designed to target and to inhibit any single gene within an organism's genome. For example, the oligonucleotides of SEQ ID NO:1 and SEQ ID NO:5 are phosphorothioate oligonucleotides complementary to the gag and rev regions of HIV-1 which inhibit HIV-1 replication, and the phosphorothioate oligonucleotide of SEQ ID NO:8 binds to the human p53 oncogene. The antisense approach is currently the only known strategy that has broad potential for precise and effective modulation of the expression of specific genes in a disease situation.
However, some antisense oligonucleotides containing phosphorothioate linkages exhibit an immunostimulatory response, causing B cell proliferation and/or an antibody response both in vitro and in vivo. This immunostimulatory response is not characteristic of all antisense oligonucleotides containing phosphorothioate linkages. For example, it is known that the phosphorothioate oligonucleotide of SEQ ID NO:8 does not induce an immunostimulatory effect.
Phosphorothioate oligonucleotide immunostimulatory effects appear to be dependent on particular sequences within the oligonucleotide but remain independent of whether the oligonucleotide is antisense, sense, or scrambled with respect to the respective target gene. Some phosphorothioate oligonucleotides induce only cell proliferation, and other phosphorothioate oligonucleotides produce no immunostimulatory effect at all. McIntyre et al. (1993) Antisense Res. Dev. 3:309-322 discloses that certain oligonucleotides can cause pronounced splenomegaly in athymic nude mice. Messina et al. (1993) Cell Immunol. 147:148-157; and Pisetsky et al. (1994) Life Sciences 54:101-107 disclose that DNA as well as structurally related synthetic oligonucleotides and polynucleotides stimulate lymphocytes, but the mechanism for this stimulation is still not fully understood. B cells are usually activated from the resting state by antigen binding to surface immunoglobulin. In mice, activation can also be modulated by physiological mediators, such as interleukin-2 (IL-2), interleukin-4 (IL-4), .gamma.-interferon, and non-physiological mitogens, such as lipopolysaccharide (LPS), Concanavalin A (con A), and pokeweed mitogen (PWM).
Certain sequence motifs or structures of oligonucleotides may play important roles in causing stimulation of murine cells. Kuramoto et al. (1992) Jpn. J. Cancer Res. 83:1128-1131 discloses that the presence of particular palindromic sequences including 5'-CG-3' motif(s) is a critical determinant in oligonucleotides for induction of natural killer cell activation and interferon production. Krieg et al. (1995) Nature 374:546-549 discloses that optimal B cell activation requires a DNA motif in which an unmethylated CpG dinucleotide is flanked by two 5'-purines and two 3'-pyrimidines.
Because of the continued need for specific treatments for diseases and inherited conditions, and the high level of specificity provided through use of antisense therapeutics capable of modulating the expression levels of targeted genes, a need exists for reducing the immunostimulatory response induced by certain phosphorothioate oligonucleotides.